The Principal Project will characterize B cell responses in patients with autoimmune disease by using a DNA-barcoding technology recently developed in the Robinson lab, termed 'antibody repertoire capture' (ARC). The approach couples DNA barcoding with next-generation sequencing to enable large-scale characterization of the paired heavy-chain (HC) and light-chain (LC) immunoglobulin genes expressed by single plasmablasts or antigen-specific B cells. Although methods exist for profiling antibodies, none are able to comprehensively characterize the, antibodies involved in an active immune response and to then bioinformatically identify those most likely to be functional i.e., those that either drive the disease or serve as identifiers of the key antigens that trigger pathogenic autoimmune responses. The scale of the sequencing datasets generated by ARC enables bioinformatic generation of phylogenetic trees of the antibody repertoire. These phylogenetic trees guide identification of clonal families of affinity-matured antibodies and thereby rational selection of key antibodies, which can then be expressed for direct analysis of their binding and functional properties. We propose to use ARC to sequence and comprehensively dissect the autoantibody responses in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), and, leveraging resources from the other ACE Projects and Cores, to test the overarching hypothesis that monoclonal autoantibodies contribute to the pathogenesis of RA and SLE by forming proinflammatory immune complexes (ICs) that dual-stimulate immune cells (by simultaneously engaging a pattern recognition receptor and either the B-cell or the Fc receptor). For instance, we hypothesize that RA-associated anti-citrullinated proteins antibodies (ACPAs) form ICs that dual-stimulate macrophages to produce TNF, and B cells to produce ACPAs; and that SLE-associated anti-nuclear antibodies (ANAs) bind nuclear antigens and thereby form ICs that dual-stimulate dendritic cells to produce IFN, and B cells to produce ANAs. In Aim 1, we will use ARC to sequence the antibody repertoires in patients with RA or SLE and identify antibody profiles that are associated with specific clinical subtypes or response to therapy. In Aim 2, we will clone and express rationally selected, affinity-matured antibodies from individuals with RA or SLE, and elucidate their autoantigen targets. In Aim 3, will characterize key RA and SLE recombinant antibodies identified in Aim 2 and uncover mechanisnis by which they contribute to autoimmune inflammation. Success would provide insights into the role of autoantibodies and the mechanisms by which they contribute to the pathogenesis of RA and SLE, and could lead to development of novel diagnostic tests and therapeutic approaches.